Light absorption detectors for high performance liquid chromatography ("HPLC"), capillary liquid chromatography (capillary LC or "CLC"), capillary electrophoresis (CE) and capillary electrochromatography ("CEC") generally include four basic components; a light source, a means for selecting range of wavelengths, a flow cell and at least one light sensor that measures the amount of light being transmitted through the flow cell. The apparatus may be disposed with parallel optical paths as a double beam instrument. The flow cell is typically in the form of a hollow tube through which a sample to be analyzed and the light are passed. Also these four basic components are typically configured to be in a predetermined arrangement with respect to each other. With these kinds of detectors, it is known that when a sample to be analyzed passes through the flow cell, the amount of light transmitted through the flow cell decreases in accordance with Beer's law.
In conventional flow cells, light is typically passed through the flow cell in one of two fashions, along the long axis of the hollow tube or perpendicular to the long axis. In either case, i.e. when the light is parallel to the long axis or introduced perpendicular thereto, the detector or light sensor output is usually expressed in terms of absorbance, which is proportional to the sample concentration and the pathlength. Thus, the longer the pathlength, the larger the detector output signal should be for a given sample concentration. For conventional flow cells, however, the light striking the lateral wall of the flow cell is partially lost due to absorption and scattering at the wall. This lost light reduces the light energy throughput of the flowcell thereby causing an increase in the noise in the output signal of the detector.
The lateral dimension or diameter of the flow cell could be increased to reduce the fraction of light striking the lateral wall, but this increases the volume of the flow cell. Frequently, however, the quantity of the sample is limited, thus the optimum cell has a minimum volume implying a small cross-section or diameter. In addition, a larger cell volume also has the effect of spreading out or dispersing the sample peak and causing a loss in chromatographic resolution. Thus, as a practical matter the foregoing effects limit the pathlength and lateral dimension of conventional flow cells.
There is described in U.S. Pat. No. 5,608,517 a flow cell having a flow passage that is coated with a polymer having an index of refraction lower than that of common chromatography solvents, e.g. water. With such a flow cell, the light being directed into the flow cell is internally reflected or piped down the length of the flow passage. It is difficult, however, with such a flow cell to obtain consistent well adhered layers of the polymer on the inner wall of the housing for the flow cell. Thus, it is possible for the polymer layer to become detached from the housing inner wall or delaminated therefrom. This delaminating effect leads to distortion of the fluid flow channel causing distortion of the optical path and the fluid flow in the flow cell, as well as creating a condition whereby the fluid could flow between the polymer and the housing.
There also is described in U.S. Pat. No. 5,184,192, owned by the assignee of the present invention, a flow cell having an inner wall formed of an amorphous fluoropolymer having a refractive index less than the refractive index of common chromatography solvents, e.g. water. Although the flow cells described therein are capable of yielding a cell with a long pathlength, the process required for making such a flow cell, and the resultant flow cell, may not be suitable for particular applications.
Moreover, the apparatuses or systems using either of these referenced flow cells must be arranged so the flow cell is precisely located with respect to the other components constituting the apparatus or system. That is, these flow cells must be precisely located in the optical path between the light source and the light sensor or detector. The criticality of the location of known flow cell(s) is the result of the need for continual, optimal alignment, which yields greater reliability and greater analytical reproducibility. Additionally, it is generally preferred to minimize the length of the flow channel between for example the chromatography column and the flow cell, which is necessarily limited by construction and design of such apparatuses or devices. Thus, even though the above described flow cells may be capable of achieving a long path length, they are typically located with respect to other components of the detection device or apparatus in the same fashion as conventional flow cells, which do not generally optimize placement from a chromatography standpoint.
A flow cell described in U.S. Pat. No. 4,867,559 includes a cladding liquid passed through a capillary in order to coat the interior bore with a low refractive index fluorocarbon. The fluorocarbon is disclosed as being a viscous, inert, immiscible, nonwetting material, such as fluorinated oil generally available under the tradenames Fluorinert.RTM. or Krytox.RTM.. This arrangement, however, is extremely unsatisfactory in practice or use. This arrangement requires a complex fluid cladding handling mechanism, including pumps and plumbing, for delivering the cladding fluid to the capillary bore, and retrieving the excess fluid after the bore has been coated. Also, interfaces and seals must be implemented, which allow delivery of the cladding fluid to the bore while not interfering with delivery of the sample that is to be delivered to the bore subsequent to the coating by the cladding fluid. Further, the delivery and withdrawal of excess cladding fluid requires careful flow calibration, as the viscosity of the fluid and the small bore of the capillary can lead to irregular coating in the capillary. This adds considerably to the complexity and expense of the flow cell.
In addition, the cladding fluid is not bonded to the interior surface of the bore, thus it must be delivered shortly before the introduction of analyte in order to prevent settling of the cladding fluid inside the bore. Further, the cladding fluid may not fully coat the interior surface of the bore, leaving gaps in the cladding that leads to scattering of the optical signal and signal attenuation. Moreover, another problem that can result is the contamination of the sample with the cladding fluid, precluding the possibility of collection of pure fractions of the sample, which could possibly interfere with a downstream analytical technique, such as mass spectrometry.